Effect of ⁶⁰Co Gamma Irradiation on Storability of Soybean Seed

MATERIALS AND METHODS

Characteristics of the soybean genotypes used for the study
The three soybean genotypes: Gmx 92-6-10, Gmx 92-5-4^E and TGX. 87^D1303 were experimental lines obtained from the Crops Research Institute, Fumesua, Ghana in June 2000. From August to November, 2000 the seeds were multiplied to raise enough materials for the study.

Genotype Gmx 92-5-4^E is yellow seeded; it takes 44 days to flower and matures in 92 days when the plant is 51 cm tall. It has seed yield potential of 1.2 t ha^-1. Seeds of Gmx 92-6-10 are green. This genotype flowers in 41 days after planting and matures in 95 days when the average plant height is 44 cm. It has seed yield of 1 t ha^-1. Genotype TGX 87^D-1303 is black seeded; it takes 35 days to flower and matures in 92 days. The seed yield potential is 0.83 t ha^-1 and has an average plant height of 78 cm (Annual Report, CRI, 1997).

Radiation dosage response studies: An amount of 250 g of seeds from each of the three soybean genotypes were dried to 10% moisture content and subjected to 0, 50, 100, 150, 200, 250 or 300 Gy of ⁶⁰Co gamma irradiation at the Ghana Atomic Energy Commission (GAEC), Kwabenya, Ghana. The irradiated seeds and their control were kept in brown paper envelopes and transported back to Kumasi the following day after irradiation and sown in 1x1.4 m sized seed boxes at the Arable Crops Research Farm of Kwame Nkrumah University of Science and Technology, Kumasi, Ghana in April 2001. Twenty seeds from each treatment dosage per genotype were drilled in rows spaced at 20 cm. The treatment combinations were replicated 4 times in a Randomized Complete Block Design (RCBD) and percentage emergence and height of all seedlings recorded ten days after planting.

Field experimentation: An amount of 2 kg of seeds from each of the three genotypes was subjected to 250 Gy of gamma rays at Kwabenya, Ghana in May 2001, after which they were transported back to Kumasi in paper envelopes. The irradiated seeds were then planted on the field two days later by drilling in rows spaced at 75 cm at the Arable Crops Research Farm of KNUST. An amount of 200 g of unirradiated seeds from each genotype was also sown similarly to serve as check. Plot size per treatment was 4x5 m. Treatments were replicated 4 times in a randomized complete block design. Karate 2.5 EC at the rate of 0.6 l ha^-1 and Thiodan 35 EC at the rate of 2 l ha^-1 were sprayed to control pre-flowering and post-flowering insect pests respectively. Weeding was done using a hoe as and when it was necessary. All M₁ plants were harvested at maturity in August 2001 and the pods dried in the sun for one week until seed moisture content was found to average 10%. The soybean pods were kept in sacks and threshed by beating the content of the sacks with sticks. Seeds of the M₁ generation were bulked together and sown again in September 2001 on the field to raise the M₂ generation.

Screening for improved storability: A total of 5,000 single M₂ plants were harvested in December, 2001 and divided at random into two groups. Plants in group one (Category A) were threshed and a total of 1,364 seeds were obtained. In group two, plants were unthreshed (Category B) and consisted of 432 seeds. Seeds and pods from individual plants in group one and two respectively were kept in brown paper envelopes and stored on laboratory wooden shelves at a temperature range of 22-25^OC and relative humidity of 30-35 % for a period of four months from January to April, 2002. Similarly, a random sample of unirradiated plants comprising 175 threshed and 152 unthreshed seeds were stored under the same conditions. At the end of the storage period, seeds from the two groups and their control (unirradiated) were drilled in the field in rows spaced at 75 cm on individual plant basis. Ten days after planting, percentage of seeds that emerged out of the total number of seeds sown from each plant was determined. Those seed lots with 80% emergence or above from each category were allowed to grow to maturity as M₃ generation and were considered as putative mutants with improved storability while seedlings from seed lots with less than 80% emergence were eliminated. The percentage emergence of the control was similarly calculated. A total of 2,000 single M₃ plants were harvested at the end of July 2002 for screening using the same screening methodologies as in the M₂ generation.

Data analysis: Data on percentage emergence and height of seedlings from the dosage response studies were subjected to analysis of variance using the 6th Edition of Genstat Statistical Software. Treatment means were separated using the least significant difference method. Variability in storability of variants created through mutagenesis in the M₂ and M₃ generations was determined using coefficient of variation of germination. Improvement in storability of variants was calculated as gain in selection expressed as the difference in mean percentage germination of the M₃ and M₂ populations (M₃-M₂). Dividing the means of the M₃ or M₂ population by the means of their control (unirradiated seeds), or the mean of M₃ population by the mean of the M₂ also gave an indication of the amount of improvement in storability created by induced mutation.

RESULTS AND DISCUSSION

Percentage emergence and seedling height: Percentage emergence and seedling height of irradiated seeds varied significantly among the dosages and genotypes as shown in Table 1 and 2. These traits decreased as the radiation dosage increased. The 250 Gy dose caused about 50% reduction in emergence and seedling height (LD₅₀) and so this dosage was chosen to treat the bulk of the seeds.

Variability in storability created through mutagenesis: For each genotype the total number of variants that recorded germination percentage in the range of 81-100% were selected and considered as putative mutants with improved storability. This was expressed as a percentage of the total number of plants screened. Screening by storing threshed seeds (Category A) produced 109, 55 and 63 putative mutants from 491, 312 and 561 single plants in Gmx 92-6-10, Gmx 92-5-4^E and TGX 87^D-1303 respectively (Table 3).

Table 1:	Percentage reduction in emergence of irradiated seeds
CV % = 5.0, LSD (0.05): Dosage means = 1.52; Genotype means = 1.08; Dosage x Genotype = 2.64

Table 2:	Percentage reduction in height of irradiated seeds
CV % = 4.7, LSD (0.05): Dosage means = 1.31; Genotype means = 0.93; Dosage x Genotype = 2.27

These values represented 22%, 17.6 and 11.2 respectively of the total M₂ plants screened from the genotypes. For Category B, 41, 30 and 15 putative mutants out of 162, 129 and 141 M₂ plants from Gmx 92–6-10, Gmx 92-5-4^E and TGX 87^D-1303, respectively were selected representing 25.3%, 23.3% and 10.6% of the total M₂ plants screened.

Genotype Gmx 92-6-10 produced the highest proportion of putative mutants in the M₂ generation. The differences observed with respect to the proportion of variants, which showed improved storability, among other factors could be attributable to differences in the genetic make-up of the genotypes. This means that storability of soybean seeds significantly varies among genotypes and is genetically determined. This finding is in agreement with Thseng et al. (1997) who made a similar observation. Out of 345, 335 and 326 single plants-screened from Category A, 175, 127 and 112 putative mutants were selected respectively from Gmx 92-6-10, Gmx 92-5-4^E and TGX 87^D-1303. This represented 51, 38 and 32% of the total M₃ plants screened (Table 4). Similarly in Category B, 63, 43 and 42% of the total plants screened from Gmx 926-10, Gmx 92-5-4^E and TGX 87^D-1303 were selected.

Table 3:	Response of variants of three soybean genotypes to screening in the M2 generation
CV% = 5.9, LSD (0.05): Genotypes = 1.1; Screening Methods = 0.9; Genotype x Screening Method = 1.6, Rad = irradiated seeds, Cont = Control/unirradiated seeds

Table 4:	Response of three soybean genotypes to screening in the M3 generation
CV% = 8.3, Genotypes = 2.06; Screening Methods = 1.68; Genotypes x Screening Methods = 2.90, Rad = irradiated seeds, Cont = Control/unirradiated seeds

Table 5:	Coefficient of variation (cv) of germination of soybean variants stored under two conditions
Rad = irradiated seeds, Cont = Control/unirradiated seeds

Table 6:	Improvement in storability of Putative Mutants
Rad = irradiated seeds, Cont = Control/unirradiated seeds

Generally, the frequency distribution of storability of the irradiated seeds was skewed to the right whereas that of the control was largely skewed to the left and this implies that some putative mutants with improved storability had been produced through induced mutation.

Coefficient of variation: Calculating coefficient of variation of germination indicated the extent of variation created by irradiation. Generally, the M₂ variability was higher than that of M₃ and those of the controls as shown in Table 5. The reduced variability in the M₃ generation could be attributed to the effectiveness of selection at the M₂ generation and also the fact that some putative mutants with improved storability had been produced and that plants in the M₃ population were beginning to approach uniformity with respect to the altered trait. Category A created higher variation than Category B. In selection, the greater the variability, the higher the chance for selecting any desirable trait.

The high variation associated with Category A resulted in high gain in selection of variants screened by this method (Table 6). Thus, the highest gain in selection of variants from genotype TGX 87^D-1303 is due to the high variation created in this genotype in the M₂ generation.

Improvement in storability of mutants: Differences were observed among genotypes with respect to improvement in storability of mutants. Genotype Gmx 92-6-10 produced mutants with the highest improvement in storability particularly at the M₂ generation (Table 6) because it responded the greatest to irradiation. At the M₃ generation, however, TGX 87^D-1303 produced the highest gain in selection whilst Gmx 92-5-4^E gave the least. Genotype TGX 87^D-1303 in the M₂ generation, produced some variants whose storability were less than the unirradiated seeds (control) and this explains why the genotype had negative improvement in the M₂ generation.

CONCLUSIONS

Dosage response studies using relative reduction in emergence and seedling height revealed that the most appropriate dosage for inducing variation in the three genotypes of soybean is 250 Gy of gamma rays. Variability created in the M₂ generation was high compared to that in the M₃ generation and the control. The reduced variation in the M₃ generation could be attributed to the effectiveness of selection in the M₂ generation and the fact that some putative mutants with improved storability had been produced. Genotype Gmx 92-6-10 produced the largest proportion of putative mutants with improved storability in M₂ whilst TGX 87^D-1303 produced the highest gain in selection at the M₃ generation.